Mechanisms of ZNF407 in regulating cutaneous melanoma by inhibiting TGF-β/Smad pathway
Objective To study the relative expression and function of the nuclear transcription factor ZNF407 in melanoma and its influence and mechanism on the migration and invasion.Methods From January 2015 to January 2021,42 cases of cutaneous malig-nant melanoma and 20 cases of pigmented nevus in Department of Dermatology,Chongqing Traditional Chinese Medicine Hospital were collected.We examined the expression of ZNF407 in melanoma cell lines and tissues through semi-quantitative polymerase chain Reaction(qRT-PCR)and westernblot.Pearson correlation analysis detection was used to analysis mRNA expression of ZNF407 and TGF-β-1.Melanoma cell line A375 was set as target cells,pcDNA3.1(+)-Flag-ZNF407(experimental group)and pcDNA3.1(+)-Flag-Vector(negative control group)were transfected,respectively.The non transfected group was the set as blank control group.Transwell assay was used to detect the effects of celluar migration and invasion;the effects of ZNF407 on the expression levels of epithelial mesenchymal transition(EMT)related mRNA were detected by semi quantitative PCR and real-time quantitative PCR.Westernblots were used to ex-plore the mechanisms of ZNF407 in epithelial-mesenchymal transition and the mechanisms of ZNF407 on TGF/β signaling pathway.Results ZNF407 protein and mRNA were downregulated in human malignant melanoma tissues compared with normal tissues and its mRNA was down-regulated in melanoma cell lines(P<0.001).Compared with the control group,transwell migration assay showed that the number of cells in the ZNF407 group(116±17)was lower than that in the control group(265±27,P<0.01)and blank control group(259±28,P<0.01).Transwell invasion assay showed:The number of invated cells in the experimental group(82±14)was lower than that in the control group(215±31,P<0.01)and blank control group(209±32,P<0.01).Westernblot showed that overexpression of ZNF407 resulted in downregulation of protein expression of Vimentin,N-cadherin,Twist,and MMP7;The protein expression of E-cadherin was upregulated(P<0.001);RT-PCR and qRT-PCR showed that over-expression of ZNF407 mRNA resulted in downregulation of stem cell markers ABCG2,Snail2,and OCT4 mRNA expression in the experimental group(P<0.001).Correlation analysis showed that ZNF407 and TGF-β1 mRNA expression was negatively correlated in melanoma tissues(P=0.033,r=-0.714).And over-expressed of ZNF407 inhibited cell migration and invation by downregulating of TGF-β-1 pathways and its downstream target proteins p-Smad1、p-Smad2、p-Smad3、p-Smad4 and c-Myc.Conclusion The expression of ZNF407 protein and mRNA was down-regulated in melanoma,over-expressed of ZNF407 expression could inhibit the cell migration and invasion of melanoma cell lines.Overexpression of ZNF407 could inhibit the ex-pression of TGF-β/Smad signal and its downstream of the pathway,and it could be acted as an important tumor suppressor gene in primary melanoma.
MelanomaZNF407Gene expressionMigration and invationTGF-β/Smad
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